PKG and PKC Are Down-Regulated during Cardiomyocyte Differentiation from Embryonic Stem Cells: Manipulation of These Pathways Enhances Cardiomyocyte Production

Understanding signal transduction mechanisms that drive differentiation of adult or embryonic stem cells (ESCs) is imperative if they are to be used to cure disease. While the list of signaling pathways regulating stem cell differentiation is growing, it is far from complete. Indentifying regulatory mechanisms and timecourse commitment to cell lineages is needed for generating pure populations terminally differentiated cell types, and in ESCs, suppression of teratoma formation. To this end, we investigated specific signaling mechanisms involved in cardiomyogenesis, followed by manipulation of these pathways to enhance differentiation of ESCs into cardiomyocytes. Subjecting nascent ESC-derived cardiomyocytes to a proteomics assay, we found that cardiomyogenesis is influenced by up- and down-regulation of a number of kinases, one of which, cGMP-dependent protein kinase (PKG), is markedly down-regulated during differentiation. Delving further, we found that manipulating the PKG pathway using PKG-specific inhibitors produced significantly more cardiomyocytes from ESCs when compared to ESCs left to differentiate without inhibitors. In addition, we found combinatorial effects when culturing ESCs in inhibitors to PKG and PKC isotypes. Consequently, we have generated a novel hypothesis: Down-regulation of PKG and specific PKC pathways are necessary for cardiomyogenesis, and when manipulated, these pathways produce significantly more cardiomyocytes than untreated ESCs

miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation

AbstractMicroRNAs are small non-coding RNAs that regulate protein expression by binding 3′UTRs of target mRNAs, thereby inhibiting translation. Similar to siRNAs, miRNAs are cleaved by Dicer. Mouse and ES cell Dicer mutants demonstrate that microRNAs are necessary for embryonic development and cellular differentiation. However, technical obstacles and the relative infancy of this field have resulted in few data on the functional significance of individual microRNAs. We present evidence that miR-17 family members, miR-17-5p, miR-20a, miR-93, and miR-106a, are differentially expressed in developing mouse embryos and function to control differentiation of stem cells. Specifically, miR-93 localizes to differentiating primitive endoderm and trophectoderm of the blastocyst. We also observe high miR-93 and miR-17-5p expression within the mesoderm of gastrulating embryos. Using an ES cell model system, we demonstrate that modulation of these miRNAs delays or enhances differentiation into the germ layers. Additionally, we demonstrate that these miRNAs regulate STAT3 mRNA in vitro. We suggest that STAT3, a known ES cell regulator, is one target mRNA responsible for the effects of these miRNAs on cellular differentiation

Stress hormone epinephrine enhances adipogenesis in murine embryonic stem cells by up-regulating the neuropeptide Y system.

Prenatal stress, psychologically and metabolically, increases the risk of obesity and diabetes in the progeny. However, the mechanisms of the pathogenesis remain unknown. In adult mice, stress activates NPY and its Y2R in a glucocorticoid-dependent manner in the abdominal fat. This increased adipogenesis and angiogenesis, leading to abdominal obesity and metabolic syndrome which were inhibited by intra-fat Y2R inactivation. To determine whether stress elevates NPY system and accelerates adipogenic potential of embryo, here we "stressed" murine embryonic stem cells (mESCs) in vitro with epinephrine (EPI) during their adipogenic differentiation. EPI was added during the commitment stage together with insulin, and followed by dexamethasone in the standard adipogenic differentiation medium. Undifferentiated embryonic bodies (EBs) showed no detectable expression of NPY. EPI markedly up-regulated the expression NPY and the Y1R at the commitment stage, followed by increased Y2R mRNA at the late of the commitment stage and the differentiation stage. EPI significantly increased EB cells proliferation and expression of the preadipocyte marker Pref-1 at the commitment stage. EPI also accelerated and amplified adipogenic differentiation detected by increasing the adipocyte markers FABP4 and PPARγ mRNAs and Oil-red O-staining at the end of the differentiation stage. EPI-induced adipogenesis was completely prevented by antagonists of the NPY receptors (Y1R+Y2R+Y5R), indicating that it was mediated by the NPY system in mESC's. Taken together, these data suggest that stress may play an important role in programming ESCs for accelerated adipogenesis by altering the stress induced hormonal regulation of the NPY system